Highly specific surface for biological reactions having an exposed ethylenic double bond, process of using the surface, and method for assaying for a molecule using the surface

ABSTRACT

The present invention relates especially to a highly specific surface for biological reactions, characterized in that it contains a support having at the surface at least one essentially compact layer of an organic compound having, outside the layer, an exposed group containing an ethylenic double bond having affinity for one type of molecule with biological activity under certain reaction conditions, the other elements of the layer being essentially inaccessible for the said molecules under the said reaction conditions.

BACKGROUND OF THE INVENTION

The present invention relates especially to very highly specificsurfaces which can be used in biology, as well as to their applicationsand to processes for preparing them.

The very high specificity and the very high selectivity of certainbiological reactions, especially antigen/antibody reactions, DNA or RNAhybridization reactions, interprotein or avidin/streptavidin/biotin typereactions, as well as reactions of ligands and their receptors, havebeen known for a long time.

It is now known how to take advantage of these specificities, especiallyin order to detect the presence or the absence of one of the elements ofthe reaction pair in a sample or alternatively for separating one of theelements of the pair from a more complex medium.

However, when it is desired to detect the presence of a molecule at avery low concentration in a very complex medium, currently knownprocesses sometimes give very unpredictable results given especially theproblem of background noise which occurs during the separation and/ordetection stages.

Consequently, what will be called hereinafter "molecular fishing", thatis to say the possibility of being able to detect each of the search formolecules when they are at very low concentrations, has so far not beenpossible.

By way of example, the analysis of a DNA sample requires the use of aso-called "hybridization" probe corresponding to the sequencecomplementary to the desired sequence. Under these conditions, theproblem posed is to isolate the hybrid from the medium and to detect,with a good signal/noise (S/N) ratio, the possibly reduced number ofpositive reactions.

Consequently, an intermediate stage intended to amplify the soughtsequence is now used in most cases, for example using the PCR method oramplification methods which lead to the same results; under theseconditions, the concentration of the sequence to be determined isincreased in the sample and its detection is obviously much easier.

However, the amplification stage is sensitive to contaminants and leadsto errors which are specific to it.

It would therefore be preferable, as far as possible, to be able todetect the presence of the nucleic acid sequence without anamplification phase.

It has been proposed to use, in order to detect the specifichybridization reaction, an intermediate stage of anchoring thehybridization product on a solid surface having certain specificities.For example, it is possible to use certain pretreated surfaces whichmake it possible to attach certain proteins or DNA, whether it has beenmodified or not.

Such surfaces are commercially available (Coralink, Costar, Estapor,Bangs, Dynal for example) in the form of beads or wells having at theirsurface COOH, NH₂ or OH groups for example.

It has also been proposed, in order to obtain such groups, to use anintermediate stage having a vinyl group which is then oxidized so as tohave COOH or OH groups (U.S. Pat. No. 4,539,061 and EP 435 785).

It is then possible to functionalize the DNA with a reactive group, forexample an amine, and to carry out a reaction with these surfaces. Thesemethods require, however, a specific functionalization of the DNA to beattached.

A technique has also been described permitting the anchoring withoutprior treatment of the DNA. This process consists in reacting the freephosphate of the 5' end of the molecule with a secondary amine (NHCoralink surface).

It is also possible to attach the DNA to a group or a protein P₀ inorder to cause it to react with a surface coated with a group or aprotein P₁, which is capable of reacting specifically with P₀. The P₀/P₁ pair may be a pair of the following type: biotin/streptavidin ordigoxigenin/antibody directed against digoxigenin (anti-DIG)-forexample.

Such surfaces are, however, in most cases insufficiently specific (V.Lund et al., Nucl. Acids Res., 16, 1861 (1988)). Thus, the presence ofunwanted, even weak, interactions of the nonspecific adsorption typeleads to efficient adsorptions for long molecules capable of forming,with the solid, a large number of points of weak interaction. Thesesurfaces lead to potential applications which lack sensitivity and/orwith a high level of background noise in the case of a small number ofmolecules to be fished out. Furthermore, some of these surfaces have ahigh level of unwanted fluorescence which is potentially disruptiveduring the detection phase.

As regards the detection itself, in particular for the detection of DNA,French Patent 78 10975 describes a process coupling the probe with anenzyme which allows revealing by means of a chromogenic substrate. Itis, in addition, possible to quantify the reaction by a colorimetricmeasurement.

Such a technique is, however, not directly adapted to the detection oftraces; consequently, here too, it should be preceded in most cases byan amplification stage for the desired quantity of nucleic acid, forexample by the PCR method.

This so-called process of detection "by a cold probe" was developed inorder to avoid the use of radioactive markers which yield results closein terms of sensitivity but which obviously present handling theproblems, given the presence of radioactive products and problems oflong revealing times if a high sensitivity is desired.

For certain specific applications, especially methods derived from exvivo imaging, there has been proposed a direct method for observing thereaction by coupling the product of the hybridization to microbeads,especially PMMA, suitably treated chemically at their surface. Themethod is based on the direct identification, under a scanning electronmicroscope, of the presence of these microbeads with a typical diameterof 60 nm, and is furthermore based on known but insufficiently specifictechniques for anchoring on solids, as described above.

The above techniques are obviously not limited to the detection ofnucleic acids. In the same spirit, the detection of antibodies has beenproposed. They are ELISA type tests which will not be described here andwhich, to summarize, make it possible to couple the presence of anantibody to an associated anchoring of a molecule of antigen on a solid.Again, the problems of specificity and unwanted reactions exist. Thedetection phase can then be based on a coupling to a chromogenicreaction having its own problems of sensitivity.

In summary, the prior art methods or combinations thereof have a numberof disadvantages, in particular:

either of being potentially dangerous because of the use of radioactiveproducts,

or of requiring revealing times which are too long,

or of being disrupted by specific problems at the level of theamplification phase,

or of requiring solid surfaces which are not sufficiently specific

or of being too weakly sensitive,

or, finally, of requiring, in addition to the phase for attachment to asolid, the use of an electron microscope which is obviously not veryconvenient.

Finally, in most cases, the known processes do not make it possible torecognize, on a given molecule, the specific position of the desiredunit. Now, this type of recognition is important when it is desired toperform mapping, in particular within the framework of genome mapping,it is desired to recognize, in a first instance, the approximate spatialposition relative to one end of the molecule of a given gene on a DNA oran RNA.

SUMMARY OF THE INVENTION

The present invention, which proposes to overcome the disadvantages ofthe prior processes, is based on the use of very highly specificsurfaces which, during their use, lead to excessively limited backgroundnoise, in particular because of the fact that they eliminate unwantedattachments.

BRIEF DESCRIPTION OF THE DRAWINGS

The descriptions that follow will be made with reference to theaccompanying Figures in which:

FIG. 1 schematically represents the detection of a pathogen in afluorescent DNA molecule by hybridization with an anchor molecule;

FIG. 2 schematically represents genetic mapping by extension of DNA andusing a marker DNA;

FIG. 3 schematically represents the detection of an immunologicalreaction (ELISA) by means of a "flag" molecule: a fluorescent DNA usedas reaction marker;

FIG. 4 is a fluorescence micrograph showing the extension of λ phage DNAby the progression of the meniscus, DNA molecules in solution stretchedby evaporation flow parallel to the meniscus can be seen on the left,DNA molecules in the open air after being stretched perpendicularly tothe meniscus can be seen on the right;

FIGS. 5(a) and 5(b) are fluorescence micrographs showing, respectively,a DNA labeled with digoxigenin (DIG) on a surface coated with anti-DIGand stretched by the meniscus, and, as control, an unlabeled DNA on ananti-DIG surface; the very high specificity of the surfaces and theabsence of nonspecific anchoring will be noted; and

FIG. 6 is a fluorescence micrograph showing a conventional commercialsurface, such as NUNC, the very high fluorescence inhomogeneities, whichrender these surfaces impossible to use for the fluorescent detection ofa single molecule should be noted.

DETAILED DESCRIPTION OF THE INVENTION

More particularly, the present invention relates to a highly specificsurface for biological reactions, characterized in that it contains asupport having at the surface at least one essentially compact layer ofan organic compound having, outside the layer, an exposed groupcontaining an ethyl chic double bond, especially a vinyl group, havingaffinity for one type of molecule with biological activity under certainreaction conditions, especially of pH or ionic content, the otherelements of the layer being essentially inaccessible for the saidmolecules under the said reaction conditions.

By "affinity", there should be understood here both a chemicalreactivity and an adsorption of any type, this under conditions forattachment of the said biological molecules.

By "support", it is intended to designate both a solid support and asupport consisting of a nonsolid element such as a liquid or gaseousparticle having, especially, a compact layer as described above.

The surface is "essentially compact", that is to say that it limits theaccess of the molecule with biological activity to the inner layersand/or to the support, it being understood that coating defects of thesurface can be tolerated.

These highly specific surfaces for biological reactions, contain asupport having at the surface groups with a double bond, especiallyvinyl (--CH═CH₂, hereinafter C═C surfaces) which are accessible to thesolution. They are capable of directly anchoring molecules of biologicalinterest (DNA, RNA, PNA, proteins, lipids, saccharides) under certainconditions of pH or ionic content of the medium. In particular, thesesurfaces do not require specific chemical modification either of thesurface or of the biological molecules to be anchored. There are nodocuments mentioning such a use of a surface with vinyl groups.

By "anchoring", there should be understood here an attachment bycovalent linkage resulting from a chemical reactivity, or alternativelya noncovalent linkage resulting from physicochemical interactions suchas an adsorption of any type, these under conditions of pH or ionicstrength of the medium for the attachment of the said biologicalmolecules.

The surfaces according to the present invention can be obtained usingvarious processes. There may be mentioned by way of example:

(A) an optionally branched carbon-containing polymer layer, at least 1nm thick, having:

groups containing an ethylenic double bond,

the remainder of the layer consisting of hydro- or fluorocarbon groups;

(B) surfaces obtained by depositing or anchoring on a solid one or moremolecular layers, the latter can be obtained by the formation ofsuccessive layers attached by noncovalent bonds of the Langmuir-Blodgettfilm type, or by molecular self assembly, this permitting the formationof a layer attached by a covalent bond.

In the first case, the surface can be obtained by polymerization of atleast one monomer generating at the surface of the polymer the saidgroup containing an ethylenic double bond, or alternatively by partialdepolymerization of the surface of a polymer so as to generate the saidgroup, or alternatively by deposition of polymer.

In this process, the polymer formed has vinyl bonds like a polyenederivative, especially surfaces of the synthetic rubber type, such aspolybutadiene, polyisoprene or natural rubber.

In the second case, the highly specific surface for biological reactionsaccording to the present invention contains:

on a support, a substantially monomolecular and compact layer of anorganic compound of elongated structure having at least:

an attachment group having affinity for the support, and

an exposed group containing an ethylenic double bond, having little orno affinity for the said support and the said attachment group under theattachment conditions, but having affinity for one type of biologicalmolecule.

In order to obtain an essentially compact layer, the various organiccompounds are preferably capable of reacting with each other besides theexposed group so as to create cross-linkages; an "essentially" compactmonomolecular layer is thus obtained by virtue of which the supportbecomes inaccessible or barely accessible for unwanted reactions.

Preferably, the organic compound has an attachment group at one end andan exposed group at the other end. It is of course possible to considervarious embodiments in which, for example, the attachment group would besituated in the middle of the molecule, the latter having an exposedgroup at each of its ends.

The surfaces can be analysed according to:

a) the support,

b) the molecule having an exposed group and an attachment group on thesupport,

c) the interaction between the support and the said molecule ensuringthe attachment.

The attachment can first of all be of the noncovalent type, especiallyof the hydrophilic/hydrophilic and hydrophobic/hydrophobic type, as inLangmuir-Blodgett films (K. B. Blodgett, J. Am. Chem. Soc. 57, 1007(1935) and U.S. Pat. No. 5,102,798).

In this case, the attachment group will be either hydrophilic orhydrophobic, especially alkyl or haloalkyl groups such as CH₃, CF₃,CHF₃, CH₂ F.

The attachment can also be of the covalent type, the attachment groupwill, in this case, react chemically with the support.

Certain surfaces of similar structure have already been mentioned in theelectronic field, especially when the attachments are covalent, L.Netzer and J. Sagiv, J. Am. Chem. Soc. 105, 674 (1983) and U.S. Pat. No.4,539,061.

Persons skilled in the art have available a wide range of groups. By wayof nonlimiting example, there may be mentioned groups of the metalalkoxide type, such as silane, silane chloride, ethoxysilane,methoxysilane.

The attachment group is obviously chosen as a function of the supportused. The support according to the invention may consist, at least atthe surface, of a polymer, a metal, a metal oxide, a semiconductorelement or an oxide of a semiconductor element such as a silicon oxideor one combination thereof. Glass and surface-oxidized silicon can bementioned in particular.

Among the attachment groups, there should be mentioned more particularlythe groups of the metal alkoxide type such as silane, chlorosilane,silanol, methoxysilane, ethoxysilane, silazane, phosphate, hydroxyl,hydrazide, hydrazine, amine, amide, diazonium, pyridine, sulfate,sulfonic, carboxylic, boronic, halogen, acid halide, aldehyde.

Most particularly, as attachment group, the use of groups capable ofcross-reacting with an adjacent equivalent group to give cross-linkages,will be preferred; for example, this will be derivatives of the silanetype, especially dichlorosilane, trichlorosilane, dimethoxysilane,trimethoxysilane, diethoxysilane and triethoxysilane.

These cross-linkages can also be performed at any point within the depthof the monolayer, by polymerizing it by means of reactive groups whichmay be present in the chain between the site of attachment and theexposed group. Thus, the diacetylenic groups are known to allow a uni-or two-dimensional polymerization of the monolayer.

The choice of the attachment group will obviously depend on the natureof the support, the silane type groups are quite suitable for covalentbonding on glass and silica.

Preferably, the chains linking the exposed group to the attachment groupare chains containing at least 1 carbon atom, preferably more than 6 andin general from 3 to 30 carbon atoms. When there is formation of a sidecoupling inside the layer, whether by ionic, coordination or covalentcoupling, highly ordered layers are obtained by self-assembly, even ifthe initial surface has only a limited number of active anchoring sitescompared with the number of molecules obtained in a compact monolayer.

Known techniques for surface functionalization using silane derivatives,for example: Si--OH+Cl₃ --Si--R--CH═CH₂ gives Si--O--Si--R--CH═CH₂, Rconsisting for example of (CH₂)₄, can be advantageously used in the caseof glass or silica. Such a reaction is known in the literature, with theuse of ultrapure solvents. The reaction leads to a layer of moleculeshaving their C═C end at the surface exposed outside.

Within the framework of the production of a highly specific surface, thepresent invention also relates in the context of reactions for graftingmolecules with a C═C double bond, to the use of a gaseous phase makingit possible to avoid the use of solvents.

In the case of gold, the latter being optionally in the form of a thinlayer on a substrate, known techniques for surface functionalization usethiol derivatives, for example: Au+HS--R--CH═CH₂ gives Au--S--R--CH═CH₂,R consisting for example of (CH₂)₄. Such a reaction is described haliquid medium and leads, like the preceding trichlorosilane-silicareaction, to a lawn of molecules layer having their C═C end at thesurface exposed outside.

Of course, the term "support" also covers both a single surface such asa slide, but also particles, whether silica powder or polymer beads, andalso any forms such as bar, fiber or structured support, which canmoreover be made magnetic, fluorescent or colored, as is known invarious assay technologies.

Preferably, the support will be chosen in order not to be fluorescent orbarely fluorescent when the detection will be carried out byfluorescence.

The surfaces obtained according to modes (A) or (B) above-have a highspecificity by virtue of the presence of specific reactive sites comingfrom the exposed groups or from the attached molecule.

In addition, the surfaces obtained according to modes (A) or (B) havethe following unexpected and remarkable characteristics:

(i) a specific and highly pH-dependent anchoring of the DNA by its endswithout requiring specific functionalization of the molecule,accompanied by a very low level of nonspecific interactions;

(ii) the possibility of anchoring on them proteins and other moleculesof biological interest, without special chemical modification;

(iii) the possibility of preparing surfaces which are specific towardsan antigen (for example digoxigenin) or a ligand (for example biotin);

(iv) a very low intrinsic fluorescence level, when required, afluorescence background noise, (with a typical area of 100×100 μm) whichis lower than the fluorescence signal of a single molecule to bedetected;

(v) the possibility of detecting isolated molecules with an S/N ratioindependent of the number of molecules, which is possible by virtue ofvarious techniques with a high S/N ratio which are described below andwhich are based on identifying the presence of a macroscopic markerhaving a weak nonspecific interaction with the surface.

The surfaces thus obtained are preferably coated with a molecule withbiological activity chosen from:

proteins,

nucleic acids

lipids

polysaccharides and derivatives thereof.

Among the proteins, there should be mentioned antigens and antibodies,ligands, receptors, but also products of the avidin or streptavidintype, as well as the derivatives of these compounds.

Among the RNAs and DNAs, there should also be mentioned the α, βderivatives as well as the thio derivatives and the mixed compounds suchas PNAs.

Mixed compounds such as glycopeptides and lipopolysaccharides forexample, or alternatively other elements such as viruses, cells inparticular, or chemical compounds such as biotin, can also be attached.

The attachment of the biological molecules may be covalent ornoncovalent, for example by adsorption, hydrogen bonding, hydrophobic orionic interactions for example, in which case it will be possible toadvantageously carry out cross-linking between the grafted molecules byknown methods ("Chemistry of Protein Conjugation and Cross-linking", S.C. Wong, CRC Press (1991)) and this in order to enhance their cohesion.

With an exposed group containing a --CH═CH₂ radical which will be calledhereinafter "C═C surface" or "surface with ethylenic bond", a directanchoring, in particular of DNA or proteins is possible. Within theframework of the present invention, it has been demonstrated that thesesurfaces have a reactivity which is highly pH-dependent. Thischaracteristic makes it possible to anchor the nucleic acids or theproteins, especially by their end(s), using a determined pH region andoften with a reaction rate which can be controlled by the pH.

Thus, for DNA at pH 5.5, the anchoring reaction is complete in one hour(if it is not limited by diffusion) and occurs via the ends. At pH 8 onthe other hand, the attachment is very low (reaction rate of 5 to 6orders of magnitude smaller). This pH dependent attachment effectspecific for the ends, is an improvement compared with the othersurfaces which require functionalization of the DNA (biotin, DIG, NHS,and the like) or specific reagents (carbodiimide, dimethyl pimelidate)which form a peptide or phosphorimide linkage between --NH₂ and --COOHor --POOH.

The surfaces according to the invention can anchor proteins directly(protein A, anti-DIG, antibodies, streptavidin, and the like). It hasbeen observed that (i) the activity of the molecule can be preserved and(ii) that the reactivity of the prepared surface (initially C═C) iscompletely occulted in favor of the sole reactivity of the molecule ofinterest. It is therefore possible, starting with a relatively highinitial reactivity, to pass to a surface having a very highly specificreactivity, for example that of specific sites on a protein.

By anchoring a specific antibody on the surface (for example anti-DIG),a surface is created whose reactivity is limited to the antigen (forexample the DIG group). This indicates that the initial chemical groupshave all been occulted by the antibodies anchored.

It is also possible to anchor onto the reactive (chemically orbiochemically) surfaces other molecules with biological activity,especially viruses or other components: membranes, membrane receptors,polysaccharides, PNA, in particular.

It is also possible to attach the product of a reaction of biologicalinterest (for example PCR) onto the prepared surfaces.

The present invention also relates to the surfaces obtained using theprocesses according to the present invention and all processes usingthis type of surface, whether they are processes permitting thedetection and/or the quantification of biological molecules, but alsothe separation of certain biological molecules, especially a sampleusing antigen/antibody and/or DNA, DNA/RNA coupling techniques.

The present invention also relates to processes for preparing highlyspecific surfaces for biological reactions as described above for theproduction of layers according to (A) and (B) and, in particular, theprocess characterized in that:

a substantially monomolecular and compact layer of an organic compoundof elongated structure having at least:

an attachment group having an affinity for the support, and

an exposed group containing an ethylenic double bond having no or littleaffinity for the said support and the attachment group under theattachment conditions, but having an affinity for one type of biologicalmolecule, is attached onto a support.

The present invention also relates to the applications of the treatedsurfaces to the detection of isolated molecules by means of specificreagents and of detection methods with an S/N ratio independent of thenumber of molecules detected.

Thus, in general, the present invention relates to a process fordetecting and/or assaying a molecule with biological activity in asample, characterized in that a surface as described above, on which amolecule with biological activity capable of recognizing the samplemolecule becomes attached, is used, and in that the detection or assayare carried out using a reagent, fluorescent or otherwise, which detectsthe presence of the attached molecule.

Among the reagents, there are fluorescent reagents and nonfluorescentreagents.

The fluorescent reagents contain fluorescent molecules, advantageouslychosen to be long molecules of size greater than 0.1 μm and reactingspecifically, directly or indirectly, with the pretreated surfaces. Forexample, but with no limitation being implied, a double-stranded DNAmolecule stained by means of fluorescent probes (ethidiumbromide, YOYO,fluorescent nucleotides, and the like) capable of anchoring directly ona C═C surface, or by a modification of the molecule (DIG, biotin and thelike) on a surface having complementary proteins (anti-DIG,streptavidin, and the like).

The nonfluorescent reagents consist especially of beads anchored via amolecule attached specifically, directly or indirectly, to a pretreatedsurface. By virtue of the surface treatment, these beads exhibit a weaknonspecific interaction with the surface. For example, but with nolimitation being implied, Dynal beads coated with streptavidin andanchored via a biotinylated DNA to a surface according to the presentinvention, having sites capable of reacting with the other end of theDNA molecule.

Depending on whether the desired molecule is detected directly byfluorescence or indirectly by means of the above reagents, the detectionwill be described as "direct detection" or "flag detection".

In order to limit the problems associated with the prohibitively slowreaction times, the diffusion times of the reagents towards the surfacecan be advantageously reduced using small reaction volumes. For example,but with no limitation being implied, by carrying out the reaction in avolume of a few microliters determined by the space between two surfacesof which one is treated so as to have reactive sites according to thepresent invention and the other is inert or treated so as not to havereactive sites.

The detection of the number of specific reactions which have occurredcan be carried out on a small number of molecules (typically 1 to 1000),by a low-noise macroscopic physical test requiring neither electronmicroscope nor radioactivity nor necessarily PCR.

The detection processes are capable of being carried out by personshaving only limited laboratory experience.

Depending on the reagent, two implementations of the present invention(mode X and mode Y) can be used for the low-noise macroscopic detectionof a small number of reactions for anchoring the reagent.

In the implementation of the so-called X mode of the present invention,a test of the number of specific reactions being produced is obtaineddirectly by a fluorescence technique, which makes it possible, for someembodiments of the present invention, to individually identify thenumber of sites which have reacted. In this case, the highly specificsurface is advantageously taken so as to have a very low fluorescencelevel; in particular the support should have a low fluorescence.

After anchoring the fluorescent reagent, the detection and counting ofthe possibly small number of anchoring reactions can be advantageouslycarried out by means of a fluorescence optical microscope using a lenswith a wide numeric aperture, making it possible to locate eitherdirectly with the eye, or after signal acquisition, the number ofanchored fluorescent molecules.

It is possible to advantageously carry out a scanning of the field ofobservation in order to explore a larger surface than the only fixedfield.

In the implementation of the so-called Y mode of the present invention,a bead type (for example, fluorescent, magnetic, colored) macroscopicreagent is detected.

Such a technique is derived from Manning et al. in the sense that thereaction is revealed by the presence or the absence of the microbeads.In one embodiment, a new process comprises:

(i) the use of beads with specific reactivity,

(ii) the use of beads which are not nanoscopic in size but which aresituated in the 0.1 μm-200 μm range, detectable by a macroscopictechnique, and

(iii) the absence of nonspecific reaction between beads and surface dueto the use of the product according to the present invention.

The number of these macroscopic beads each characterizing an anchoringreaction is then determined by a macroscopic physical method amongwhich, but with no limitation being implied, there may be mentioned thediffusion of light on the beads, optical microscopy and the fluorescenceof the beads.

The specificity of certain biological reactions may be limited. Thus,within the framework of the hybridization, the hybrids can be imperfect(reactions with other sites) while having a reduced number of pairingand therefore a lower quality of binding. The present invention alsocovers the possible use of a stage for testing the quality of the bondsobtained. This test makes it possible to dissociate the products whichhave paired in a weak nonspecific manner, by adsorption, hydrophobicforces, imperfect hydrogen bonds, imperfect hybridization, inparticular.

Consequently, the invention also relates, in a detection or assayprocess as described above, to a process where the product of thereaction between the molecule with biological activity and the samplemolecule is subjected to a stress in order to destroy the mismatchesbefore the detection.

This process offers, in addition to the possibility of destroying themismatched pairs, the possibility of orienting the coupling products,which facilitates the measurements or the observations.

It is thus possible to apply to the surfaces, after attachment of thecomplementary elements, a stress which may consist of the single orcombined use of:

centrifugation,

gradient of magnetic field applied to the nonfluorescent reagents taken,in this case, to include magnetizable or magnetic microbeads,

stirring,

liquid flow,

meniscus passage,

electrophoresis

temperature variation and/or temperature gradient.

The number of systems to have maintained their integrity or to havebecome destroyed is then determined by the low-noise detectiontechniques described below.

It should be noted that using the surfaces according to the presentinvention, it is possible to orient the molecules after their attachmentby at least one point by passage of the air/water meniscus, especiallyover DNA. Thus, it was observed that the passage of the air/watermeniscus over DNA in solution and anchored at the surface, resulted in auniform extension of the anchored molecules. They appear, in this case,in the open air in the form of elongated fluorescent rods. Theseelongated molecules are stable in the open air and can be observed evenafter several weeks, without showing apparent degradation.

These remarkable and unexpected observations suggest a possibility ofcounting the number of DNA molecules anchored at the surface: on the onehand, the surfaces not being highly fluorescent, the signal/noise (S/N)ratio is good, on the other hand, seeking a highly correlated object(rod shape), it is very easy to increase the S/N ratio. That is to sayto ignore the dusts, the inhomogeneities which have no special spatialcorrelation. It should be noted that in solution, the molecules in theform of a random cole fluctuate thermally thereby causing very highvariations in their fluorescence signal gathered, in general, with asmall depth of field and limiting their observation. The presentinvention also covers this alignment and immobilization technique whichtherefore allows the observation of isolated molecules with a very highS/N ratio.

It is remarkable that this ratio is independent of the number ofmolecules anchored. The S/N ratio posed by the detection of one moleculeis the same as that for 10,000. Furthermore, this stretching techniquemakes it possible to easily discriminate between molecules of varyinglengths.

It is advantageously possible to proceed to the following stages inorder to further improve the S/N ratio:

The molecule being stationary, its fluorescence signal can beintegrated.

Microscopic observation presents a reduced field (typically 100 μm×100μm with a ×100 immersion lens, N.A.=1.25). For a 1 cm² sample, scanningcan be carried out, or it is possible to envisage the use of lowermagnification lenses (×10 or ×20) but with a high numerical aperture.

The rods being always parallel, it is possible to envisage an opticalspatial filtration method in order to further increase the S/N ratio.

Other global fluorescence methods can be envisaged (EP #103426).

The linearization of the molecules is observed both within the frameworkof a chemical grafting (C═C) and in the case of immunological typelinkages (DIG/anti-DIG).

Once the surface is in the open air, the DNA molecules are stable (theymaintain their integrity even after several weeks) and fluorescent. Thisproperty can be advantageously used in order to defer the anchoringstage and the locating/counting stage for the molecules anchored, ifthis detection is done for example, but without limitation beingimplied, by fluorescence microscopy. Such a use is covered by thepresent invention.

A double (or multi) fluorescence technique can possibly be used toimprove the S/N ratio or to detect a double or multi-functionality.

It is possible to extend the air/water meniscus used here in order tostretch the molecule to other systems such as oil/water orwater/surfactant/air, in particular.

It is possible to use a dynamic orientation of the molecules in solutionanchored at one end, by electrophoresis or flow in one or moresuccessive directions, it being thereby possible for such a technique tolead to the synchronous detection of the presence of molecules in agiven direction, by analysis of the temporal variations of thefluorescence signal corresponding to a given direction (for example, butwith no limitation being implied by using a suitably arranged opticalspatial filter permitting a preferential signal to be obtained forcertain orientations of the observed molecules).

However, the observed results show that this technique, in its simplestversion (stretching in a single direction, without synchronousdetection) is much less efficient than the use of the meniscus.

The surfaces and/or the reagents and/or the detection techniquesdescribed in the present invention can be used for numerousapplications, among which, but with no limitation being implied:

the identification of one or more elements for sequencing of DNA or RNAwhich can be advantageously used for the diagnosis of pathogens orgenetic mapping;

the measurement of the size of DNA fragments which can be advantageouslyused for the genetic mapping;

the improvement of the sensitivity of the ELISA techniques with thepossibility of detecting a small number (possibly less than 1000) ofimmunological reactions.

The identification of DNA/RNA sequences can be performed first byreacting in the solution volume the DNA/RNA molecules with complementaryprobes (for example by hybridization or by means of proteins specificfor the desired segment). Two procedures are possible in this case.

In the "diagnostic" mode, the probes (the "anchors") possess a reactivegroup (DIG, biotin, and the like) capable of anchoring specifically on asurface according to the present invention (having for example asanchoring site an anti-DIG antibody or streptavidin). The detection ofthe anchoring reaction can be carried out directly by detection of thefluorescence of the DNA molecule stained by fluorescent molecules(ethidium bromide, YOYO, fluorescent nucleotides) (FIG. 1). It can alsobe carried out indirectly by detection of a "flag molecule": a reagentaccording to the present invention capable of attaching to the DNA/RNAmolecule (for example by hybridization, protein-DNA interaction, and thelike), but having no affinity for the anchoring sites of the probe.

In the "mapping" mode, the complementary probes can be directly coupledto a fluorescent reagent according to the present invention. It may befor example a single complementary DNA strand possessing bases modifiedso as to be fluorescent or a long double DNA strand stained with afluorophore A and ending with a single strand segment complementary tothe desired sequence. For different probes, fluorophores of differentcolors can be used. It is also possible to advantageously stain the DNAmolecule to which the probes have just been hybridized with afluorophore of a different color. The DNA-probe hybrid is anchored atone of its ends and stretched by one of the methods described above. Thedistance from the anchoring point to the hybridization points, orbetween the hybridization points, is determined by detecting thefluorescence of the probe, according to the methods described above(FIG. 2).

For example, with no limitation being implied, a marker DNA, of about3000 base pairs and having at one of its ends a single strand segmentcomplementary to the desired gene, is stained with a fluorophore A (forexample YOYO1). This DNA is hybridized and then ligated with thesingle-stranded DNA to be mapped, and then the latter is stained with asecond fluorophore B (POPO1) (after reacting by random priming, so as toconvert it to a double-stranded DNA). The molecule is then anchored byone of its ends (for example by DIG/anti-DIG linkage) and stretched bythe action of the meniscus. The distance between the end of the moleculeand the position of the labelled gene, which can be observed by doublefluorescence microscopy (2 colors A and B) makes it possible toestablish the position of the desired gene with a precision of the orderof 1000 base pairs (0.3 μm).

The identification of the DNA/RNA sequences can also be carried out by areaction between the desired sequence and the reactive sites of asurface according to the present invention (for example complementaryoligonucleotides or the site of reaction of a protein specific for thedesired segment). The detection of the anchoring reaction can, in thiscase, be carried out directly or indirectly (by means of a "flagmolecule") as described above.

It is well understood that the identification of the DNA/RNA sequencesaccording to the present invention can serve both for diagnosticpurposes (for example the detection of the presence or the absence of aviral or chromosomal patbogen) and for genetic mapping purposes. It canbe preceded by an amplification stage by any method, especially PCR.

Moreover, as mentioned by K. R. Allan et al. (U.S. Pat. No. 84,114), thegenetic mapping can be carried out by measuring the size of the DNAfragments. Now, the coupling between the surfaces according to thepresent invention and the novel techniques for stretching the moleculesdescribed above (in particular and advantageously the stretching by themeniscus) makes it possible to measure the length of the stretchedmolecules and this on a very small sample (a few thousandths ofmolecules).

It is for example possible, with no limitation being implied, to carryout the process in the following manner:

A DNA sample is fragmented (by means of restriction enzymes) stainedwith a fluorophore and then anchored on a surface having reactive groups(for example the C═C surfaces). The molecules are then stretched by themeniscus and the size of the stretched fragments determined byfluorescence optical microscopy with a resolution and a maximum size ofthe order of 1000 bp (0.3 μm).

The surfaces according to the present invention can be used to carry outknown processes which allow the detection and/or the quantification ofan antigen or an antibody, especially the ELISA methods using enzymaticsystems or RIA type methods using radioactive markers. Thesetechnologies will not be described in detail.

It is also possible and advantageous to use the surfaces according tothe present invention as support for the immunological reactions of anELISA process having a stage for anchoring a reagent according to thepresent invention ("flag") on one of the ELISA reagents (FIG. 3). Thedetection can naturally be carried out globally by measurement offluorescence. It is also possible to count the number of reactions; thiscan be advantageously carried out according to the detection methodsdescribed in the present invention, in particular the extension by themeniscus, and this by virtue of the low level of fluorescence andnonspecific interaction of the product of the present invention. Thisallows the detection of a small number of reactions, possibly less than1000, with an excellent S/N ratio.

It is therefore possible, by a minor modification of the sandwichedELISA methods (antibody-antigen-modified antibody, for examplebiotinylated), to graft on the surface a reagent according to thepresent invention, for example fluorescent DNA anchored on streptavidin.

All the variants of the ELISA technique apply with a much bettersensitivity. Techniques for global fluorescence measurement are alreadyused to determine the quality of the ELISA reactions. However, theprocess according to the invention allows a better detection because itis sensitive to the fluorescence signal from a single molecule.

Of course, it is possible to use these surfaces as surfaces for theattachment of at least one product of a reaction of biological interest,by way of example, with no limitation being implied, products of theamplification reaction, whether PCR or a related method and, finally, itis possible to use this type of surfaces as support for an affinitychromatography, whether preparative or for detection.

In this case, it is for example possible to graft a given population ofoligonucleotides on silica beads which will constitute the stationaryphase of a chromatographic column.

The chromatography stage should allow a characteristic specificity ofthe column with respect to the eluents, for example a DNA mixture ofwhich some have complementary sequences or sequences very close to thegrafted oligonucleotide.

The present invention finally relates to the use of the surfacesaccording to the present invention in diagnostic or separation kits.

Other characteristics and advantages of the present invention willemerge on reading the examples below.

Materials and Methods

The λ DNA and the monoclonal antibody (anti-DIG) are obtained fromBoehringer-Mannheim. The trichlorosilanes are obtained fromRoth-Sochiel. The fluorescent nucleic probes (YOYO1, YOYO3 and POPO1)are obtained from Molecular Probes. The ultraclean glass cover slips areobtained from Erie Scientific (ESCO) cover slips). The magneticparticles are obtained from Dynal. The microscope is a Diaphot invertedmicroscope from NIKKON, equipped with a Xenon lamp for epifluorescenceand a Hamamatsu intensified CCD camera for the visualization.

Surface Treatment

Glass cover slips are cleaned for one hour by UV irradiation under anoxygen atmosphere (by formation of ozone). They are then immediatelyplaced in a desiccator previously purged of traces of water by an argonstream. A volume of about 100 to 500 μl of the appropriatetrichlorosilane (H₂ C═CH--(CH₂)_(N) --SiCl₃ is introduced into thedesiccator, from which the surfaces are removed after about 12 hours(n=6) or 1 hour (n=1). Upon taking out, the surfaces are clean andnonwetting.

The cover slips thus functionalized can react with proteins. A volume of300 μl of an aqueous solution (20 μg/ml) of proteins (protein A,streptavidin, and the like) is deposited on a cover slip functionalizedwith a (H₂ C═CH--) group. This cover slip is incubated for about twohours at room temperature, then rinsed three times in ultrapure water.The surfaces thus treated are clean and wetting. The surfaces treatedwith protein A can then react with an antibody, for example an anti-DIGantibody, by incubating in a solution of 20 μg/ml of antibody.

Anchoring of Native DNA on a Double Bond Surface

A drop of 2 μl of a fluorescence-labelled λ DNA solution (YOYO1, POPO1or YOYO3, but with no specific end labelling) of varying concentrationand in different buffers (total number of molecules <10⁷) is depositedon a pretreated cover slip (H₂ C═CH) and covered with an untreated glasscover slip (diameter 18 mm). The preparation is incubated for about 1hour at room temperature in an atmosphere saturated with water vapor. Ina 0.05M MES buffer (pH=5.5), a virtually general anchoring of the DNAmolecules is observed. In contrast, in a 0.01M Tris buffer (pH=8), thereis practically no anchored molecule (ratio >10⁶). This dependence canmake it possible to control the activation/deactivation of surfaces(with respect to DNA) via the pH.

Detection of the anchoring by the action of the meniscus

By transferring the preceding preparation to a dry atmosphere, thesolution, upon evaporating, will stretch the DNA molecules anchored onthe surface, perpendicularly to the meniscus. The capillary force on theDNA molecule (a few tens of picoNewtons) is indeed sufficient tocompletely stretch the molecule (greater than the entropic elasticityforces), but too weak to break the bond between the end of the moleculeand the treated surface. The DNA having been fluorescence labelled, thestretched molecules (total length about 22 μm) can be individually andeasily observed. The anchoring between the surface and the DNA beinglimited to the ends, it was possible to stretch either DNA of λ phage,of YAC or of E. coli (total length greater than 400 μm). This DNApreparation, stretched, fluorescent and in the open air, is stable forseveral days and can be observed in a nondestructive manner, byepifluorescence (Nikkon Diaphot inverted microscope with a ×100 lens,O.N.: 1.25).

Detection of the Anchoring by Electrophoresis

An electrophoretic cell is formed by a paraffin ring (thickness of about100 μm) taken between a treated cover slip and an untreated glass coverslip between which two platinum electrodes are inserted. The assembly isrigidly held together by briefly melting the paraffin ring. The DNAsolution is introduced into this cell by capillarity through twoopenings left in the paraffin ring, then both openings in the paraffinare sealed. The incubation is carried out, as above, at roomtemperature. On applying a low voltage (few volts) between the twoplatinum electrodes, there is observed, by fluorescence, a movement ofthe free DNA molecules (a few tenths of microns per second) and anextension in the direction of the flow of the anchored molecules whichcan thus be easily and individually identified by epifluorescencemicroscopy.

Specific Anchoring and Detection

By treating the surfaces as described above with a specific-monoclonalantibody, it is possible to control their specificity very precisely.Thus, the specificity of anti-DIG treated surfaces was tested inrelation to λ DNA hybridized with an oligonucleotide complementary toone of the Cos ends and possessing a digoxigenin group (DIG) and inrelation to nonhybridized DNA. In the first case, a virtually generalextension of the anchored molecules, by the action of the meniscus, wasobserved. In the second case, only a few anchored DNA molecules (<10)were observed in the whole sample. It is therefore estimated that thespecificity of the method according to the invention is greater than10⁶.

Sensitivity of the Detection

In order to determine the sensitivity of the detection method byextension of the meniscus, 2.5 μl drops of a solution of λ DNA in 0.05MMES (pH=5.5) containing a total of 10⁵, 10⁴ and 1000 molecules, weredeposited on double bonding surfaces. The anchoring is carried out asdescribed above. The cover slips are then observed by epifluorescencemicroscopy to determine the density of the anchored molecules. Thelatter indeed corresponds to that estimated: about 4-6 DNA molecules perfield of vision (100 μm×100 μm) for a total of 10⁵ DNA molecules. Forthe lowest concentration, it was possible to observe about 10 moleculesextended by the action of the meniscus. This number is essentiallylimited by the large number of fields of vision required to cover thewhole sample (about 25,000), which makes a manual search difficult, butit can be advantageously carried out automatically and with a weakerlens, but with a larger field. In conclusion, the sensitivity of themethod according to the invention allows detection and individualCounting of less than 1000 DNA molecules.

We claim:
 1. A process for attaching a nucleic acid on a surface whichcomprises:(a) providing a surface having affinity for said nucleic acidand having exposed double bonds; (b) contacting said surface with saidnucleic acid in a fluid at a pH of about 5 to about 6 to attach one endof said nucleic acid to said surface.
 2. The process of claim 1, whereinthe fluid is a liquid.
 3. The process of claim 1, wherein the doublebonds are ethylenic double bonds.
 4. The process of claim 1, wherein theattachment of said nucleic acid to said surface is by adsorption.
 5. Theprocess of claim 1, wherein the nucleic acid is DNA.
 6. The process ofclaim 1, wherein the surface comprises a semiconductor element, an oxideof a semiconductor element, a silicon oxide, or a combination thereof.7. The product of the process of one of claims 1, 2, 3, 4, 5, or
 6. 8. Aprocess for detecting a biological molecule in a sample, comprising:(a)providing the product of claim 7; (b) contacting a sample containing abiological molecule with the product of claim 7; and (c) detecting thepresence or absence of binding of the biological molecule to theattached nucleic acid.
 9. The process according to claim 8, wherein thebiological molecule is DNA, RNA, or protein.